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A novel family with recessive von Willebrand disease due to compound heterozygosity for a splice site mutation and a missense mutation in the von Willebrand factor gene
Thrombosis Research 105 (2002) 135 – 138
Regular Article
A novel family with recessive von Willebrand disease due to compound heterozygosity for a splice site mutation and a missense mutation in the von Willebrand factor gene Giancarlo Castamana,*, Elisabetta Novellaa, Evelina Castigliaa, Jeroen C.J. Eikenboomb, Francesco Rodeghieroa a
Department of Hematology and Hemophilia and Thrombosis Center, San Bortolo Hospital, I-36100 Vicenza, Italy b Hemostasis and Thrombosis Research Center, Leiden University Medical Center, The Netherlands Received 24 September 2001; received in revised form 30 November 2001; accepted 30 November 2001 Accepting Editor: D. Prisco
Abstract We report a new family with autosomal recessive von Willebrand disease (VWD) in which the propositus was compound heterozygous for a missense mutation in exon 42 (G7085T, C2362F) and a C ! A splice site mutation in intron 13 of the von Willebrand factor (VWF) gene. The propositus had factor VIII:C 20 IU/dl, VWF antigen 5 – 7 IU/dl and ristocetin cofactor activity < 3 – 7 IU/dl. The bleeding time (BT) was markedly prolonged ( > 15 min). Haplotype analysis and mutation screening were conducted by polymerase chain reaction (PCR)based methodology. Each mutation appeared to be linked to a haplotype that was identical to the haplotype previously detected in subjects in the Veneto region carrying these mutations, suggesting a founder effect. In the propositus’ family six subjects were heterozygotes for the C2362F mutation and five were heterozygotes for the splice site mutation. None of the heterozygotes had suffered from significant bleeding. The mutation C2362F and the splice site mutation in intron 13 are commonly observed in subjects with recessive von Willebrand disease in the Veneto region. These mutations are truly recessive and cause bleeding only in the compound heterozygous or homozygous state. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: von Willebrand disease; von Willebrand factor; Inherited bleeding disorder
1. Introduction von Willebrand disease (VWD) is an inherited bleeding disorder characterized by a quantitative or qualitative defect of von Willebrand factor (VWF), an adhesive protein involved in platelet adhesion and which acts as a carrier of FVIII in blood [1]. The disease has been classified into three major groups, based on phenotypic criteria: Types 1 and 3 represent, respectively, partial and severe quantitative deficiency of VWF whereas Type 2 includes qualitative defects [1]. The molecular bases of Type 3 VWD are heterogeneous [1]. The probands may be homozygotes for
* Corresponding author. Tel.: +39-444-993679; fax: +39-444-993922. E-mail address: [email protected] (G. Castaman).
null alleles or compound heterozygotes of null alleles and missense mutations, especially when low levels of VWF are detectable [1– 3]. In some cases, it is difficult to distinguish Type 3 from recessive Type 1 on phenotypic grounds [2,3]. In a group of eight patients with recessive VWD from the Veneto region, the propositus of a single family showed compound heterozygosity for a missense mutation in exon 42 (G7085T leading to C2362F substitution in the mature subunit) and a splice site mutation C ! A at position 11/24 in the consensus sequence of intron 13 [3]. Both mutations were observed in other families with either Type 1 or Type 3 phenotype in combination with other null alleles [3]. Heterozygous subjects were asymptomatic, as usually reported for the obligatory heterozygotes of Type 3 VWD [3]. In this paper, we report a new family with autosomal recessive VWD in which the propositus was compound heterozygous for the C2362F mutation and the splice site
0049-3848/02/$ – see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 0 0 7 - 5
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G. Castaman et al. / Thrombosis Research 105 (2002) 135–138
mutation in intron 13. Haplotype analysis confirmed the possibility of a founder effect for both the mutations.
2. Materials and methods
were expressed as international units (IU), with reference to an internal plasma pool calibrated against the Second International Reference Preparation for Factor VIII/VWFrelated activities in plasma. Bleeding time (BT) was measured with the Simplate II device (General Diagnostics, Morris Plains, NJ).
2.1. Case report 2.3. DNA studies The propositus, born in 1993, was referred for investigation at age 3 after an episode of epistaxis requiring substitution treatment with FVIII/VWF concentrate. At age 2, he bled profusely after an accidental bite to the tongue. After the diagnosis, he suffered from several episodes of epistaxis, controlled by oral tranexamic acid and/or digital compression. He also had frequent easy bruising. At another hospital, factor VIII procoagulant activity (FVIII:C) was 25% and von Willebrand factor antigen (VWF:Ag) 5%. All the available family members were investigated. No bleeding episodes were observed in the relatives. The pedigree is shown in Fig. 1. 2.2. Coagulation studies Blood was anticoagulated with 3.8% sodium citrate (1:10 vol:vol) and centrifuged at 2500 g for 15 min. FVIII:C, VWF:Ag and VWF ristocetin cofactor activity (VWF:RCo) in plasma and platelets were determined as previously described [4]. All FVIII and VWF measurements
2.3.1. Haplotype analysis Genomic DNA was extracted from peripheral blood leukocytes and amplified by the polymerase chain reaction (PCR) [3] under the following conditions: denaturing at 94 °C for 4 min followed by 32 cycles of 1-min denaturing at 94 °C; 1-min annealing at 58 °C; and 2 min extension at 72 °C. Haplotypes were determined using two variable number tandem repeat (VNTR) polymorphisms located in intron 40 (indicated as VNTR I and VNTR II), RsaI polymorphism in exon 13 (G+/A 1451, Arg/His 484), RsaI polymorphism in exon 18 (A+/G 2365, Thr/Ala 789), an HphI polymorphism in exon 28 (G /A+ 4141, Ala/Thr 1381) and XmnI polymorphism in exon 45 (+ indicates presence, absence of the restriction site; nucleotide numbering from the A of the initiator ATG as + 1; amino acid numbering from the translation initiation codon), as previously described in detail [3]. VNTR I and II were examined by electrophoresis on 8% nondenaturing polyacrylamide gels and visualized under
Fig. 1. Pedigree of the family and haplotypes of the propositus and his parents. The indicated haplotypes are VNTR I and II intron 40, RsaI exon 13, RsaI exon 18, HphI exon 28, and XmnI exon 45, respectively (see Materials and Methods). The proband is indicated by an arrowhead. ND = not done.
G. Castaman et al. / Thrombosis Research 105 (2002) 135–138 Table 1 Von Willebrand factor gene mutations screened in the propositus Mutation
Exon
Method of detectiona
Splice site intron 13 4449delG 7125insC C2362F R2535a C2671Y
14 28 42 42 45 49
Eco57I sequence analysis sequence analysis Eco57I DdeI MaeIII
a
For details see Ref. [3].
UV light following ethidium bromide staining. The restriction enzyme analyses were performed directly in the PCR reaction mixture. The samples were incubated overnight at 37 °C. 2.3.2. Mutation screening DNA was amplified by PCR using oligonucleotide primers designed on the basis of the reported sequences of the VWF gene [5]. The propositus was screened for the presence of several mutations (Table 1), which were previously identified in our group of patients with recessive VWD from Veneto [3]. Digestion with the appropriate restriction enzyme was carried out directly in the PCR reaction mixture. The samples were incubated overnight at 37 °C. The pattern of digestion was visualized under UV after ethidium-bromide-stained agarose gel electrophoresis. 3. Results The propositus, subject III.2, showed marked reduction of all FVIII/VWF measurements, compared with the other
137
family members investigated (Table 2). Platelet VWF was severely reduced (VWF:Ag 2.8 IU/10 11 platelets and VWF:RCo < 3 IU/1011 platelets; normal range 17 –88 and 24 – 102 IU/1011 platelets, respectively). The BT was markedly prolonged. The father, subject II.4, showed only borderline values of VWF:RCo in plasma, whereas platelet VWF was in the normal range (not shown). The mother, subject II.5, had all FVIII/VWF measurements mildly reduced in plasma and only platelet VWF:RCo slightly reduced in platelets (18 IU/1011 platelets). BT was normal in the parents as well as in all the other family members tested. Factor VIII/VWF measurements showed a heterogeneous pattern in the remaining subjects, with subject II.6 having all the measurements reduced and the other seven with only mild VWF reduction. Only subject I.1 with the splice site mutation had normal FVIII/VWF measurements. The propositus (subject III.2; Table 2) was compound heterozygous for the splice site mutation in intron 13, inherited from the father, and the C2362F mutation, inherited from the mother (Fig. 1). All other screened mutations (Table 1) were not detected in the propositus. Apart from the propositus, five members were heterozygous for the splice site mutation and six for the C2362F mutation (Fig. 1). None of these true heterozygotes had suffered from bleeding symptoms. Haplotype analysis showed that the splice site mutation was linked to the haplotype 7, 4, +, +, , + (VNTR I and VNTR II intron 40, RsaI exon 13, RsaI exon 18, HphI exon 28 and XmnI exon 45, respectively), and the C2362F mutation to the haplotype 10, 4, +, +, , (Fig. 1). These haplotypes were similar to those previously found in association with these mutations in our population of patients with recessive VWD.
Table 2 FVIII and VWF measurements, BT, blood group, and presence of the C2362F or intron 13 splice site mutation Subjects
VIII:C (IU/dl)
VWF: Ag (IU/dl)
VWF:RCo (IU/dl)
BT (min)
Blood group
VWF gene mutation
I.1 I.2 I.3 I.4 II.1 II.2 II.4 II.5 II.6 II.7 II.8 III.1 III.2 (propositus) III.3 III.4 III.5 III.6 III.7 III.8 Normal range
149 140 128 95 116 101 66 – 102 35 38 100 120 102 18 – 21 85 82 87 114 104 126 52 – 173
109 120 123 56 53 98 70 – 77 25 34 102 117 80 5–7 45 47 35 58 56 117 47 – 165
73 130 132 41 52 97 58 – 76 29 28 80 140 78 <3–7 42 49 48 61 55 118 51 – 188
5 ND ND 4.5 6 ND 7 4 6 5.5 4.5 ND >15 ND ND 5 ND ND ND < 7.5
O B A O O O B O O A A O B O O O A A A –
splice site NO NO C2362F splice site NO splice site C2362F C2362F NO NO NO C2362F/splice site splice site splice site C2362F C2362F C2362F NO NONE
ND = not done.
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4. Discussion We have identified a new family with recessive VWD in which the propositus was compound heterozygous for a splice site mutation in intron 13 and the C2362F mutation in exon 42 of VWF gene. While these mutations have been previously identified in a propositus with recessive Type 3 VWD, this patient was not related to the present family [3]. When compared to the previous case, the present propositus had somewhat higher FVIII/VWF parameters in plasma and apparently milder clinical phenotype, so that a Type 1 VWD phenotype could be appreciated. There is no clear explanation for this finding. The only difference between the two propositi is blood group (Type B in the present, Type O in the previously reported case). However, in keeping with possible diagnosis of Type 3 VWD, platelet VWF was severely reduced in the present propositus. Each mutation was linked to a similar haplotype as in the families previously reported [3]. This strongly suggests the presence of a founder effect. As a further support to this hypothesis, these mutations have not been reported from other geographical areas. The splice site mutation leads to aberrant mRNA splicing and skipping of exon 14 [3]. Consequently, only a nonfunctional truncated VWF is coded for by this null allele. The C2362F mutation induces the loss of a cysteine in a VWF region to which no function has been assigned. Patients homozygous or hemizygous for the C2362F mutation show an increased proteolysis of the mutant C2362F VWF [6], as usually observed in typical Type 2A VWD patients [7]. In contrast, heterozygotes show a normal pattern, suggesting a protective role of the VWF coded for by the normal allele. Interestingly, patients with compound heterozygosity for this mutation and a null allele have a significant VIII:C increase after desmopressin, despite the fact that very little VWF increase is observed after infusion and BT remains markedly prolonged [4]. Heterozygosity for either the C2362F or the splice site mutation in intron 13 does not represent a risk factor for bleeding. In fact, using the pooled data from the present analysis and from the previous investigation [3], none of the
subjects who are heterozygous for C2362F mutation (n = 15) or for the splice site mutation (n = 9) suffered from bleeding, despite the occurrence of several hemostatic challenges. These observations are in keeping with the typical description of an absence of bleeding symptoms in obligatory heterozygotes for Type 3 VWD [8]. On the contrary, we observed a single patient with homozygosity for C2362F mutation and a moderate to severe bleeding history [3]. In conclusion, the C2362F mutation and the splice site mutation in intron 13 of VWF gene are commonly observed in our population of patients with recessive VWD in the Veneto region (northeast of Italy) and are likely to be the result of a founder effect.
References [1] Nichols WC, Ginsburg D. von Willebrand disease. Medicine 1997; 76:1 – 20. [2] Eikenboom JCJ, Reitsma PH, Peerlinck KMJ, Brie¨t E. Recessive inheritance of von Willebrand’s disease type I. Lancet 1993;341:982 – 6. [3] Eikenboom JCJ, Castaman G, Vos HL, Bertina RM, Rodeghiero F. Characterization of the genetic defects in recessive type 1 and type 3 von Willebrand disease patients of Italian origin. Thromb Haemost 1998;79:709 – 17. [4] Castaman G, Lattuada A, Mannucci PM, Rodeghiero F. Factor VIII:C increases after desmopressin in a subgroup of patients with autosomal recessive von Willebrand disease. Br J Haematol 1995;89:147 – 51. [5] Mancuso DJ, Tuley EA, Westfield LA, Worrall NK, Shelton-Inloes BB, Sorace JM, Alevy YG, Sadler JE. Structure of the gene for human von Willebrand factor. J Biol Chem 1989;264:19514 – 27. [6] Castaman G, Eikenboom JCJ, Lattuada A, Mannucci PM, Rodeghiero F. Heightened proteolysis of the von Willebrand factor subunit in patients with von Willebrand disease hemizygous or homozygous for the C2362F mutation. Br J Haematol 2000;108:188 – 90. [7] Zimmerman TS, Dent JA, Ruggeri ZM, Nannini LH. Subunit composition of plasma von Willebrand factor. Cleavage is present in normal individuals, increased in type IIA and IIB von Willebrand disease, but minimal in variants with aberrant structure of individuals oligomers (types IIC, IID, and IIE). J Clin Invest 1986;77:947 – 51. [8] Mannucci PM, Lattuada A, Castaman G, Lombardi R, Colibretti ML, Ciavarella N, Rodeghiero F. Heterogeneous phenotypes of platelet and plasma von Willebrand factor in obligatory heterozygotes for severe von Willebrand’s disease. Blood 1989;74:2433 – 7.
Regular Article
A novel family with recessive von Willebrand disease due to compound heterozygosity for a splice site mutation and a missense mutation in the von Willebrand factor gene Giancarlo Castamana,*, Elisabetta Novellaa, Evelina Castigliaa, Jeroen C.J. Eikenboomb, Francesco Rodeghieroa a
Department of Hematology and Hemophilia and Thrombosis Center, San Bortolo Hospital, I-36100 Vicenza, Italy b Hemostasis and Thrombosis Research Center, Leiden University Medical Center, The Netherlands Received 24 September 2001; received in revised form 30 November 2001; accepted 30 November 2001 Accepting Editor: D. Prisco
Abstract We report a new family with autosomal recessive von Willebrand disease (VWD) in which the propositus was compound heterozygous for a missense mutation in exon 42 (G7085T, C2362F) and a C ! A splice site mutation in intron 13 of the von Willebrand factor (VWF) gene. The propositus had factor VIII:C 20 IU/dl, VWF antigen 5 – 7 IU/dl and ristocetin cofactor activity < 3 – 7 IU/dl. The bleeding time (BT) was markedly prolonged ( > 15 min). Haplotype analysis and mutation screening were conducted by polymerase chain reaction (PCR)based methodology. Each mutation appeared to be linked to a haplotype that was identical to the haplotype previously detected in subjects in the Veneto region carrying these mutations, suggesting a founder effect. In the propositus’ family six subjects were heterozygotes for the C2362F mutation and five were heterozygotes for the splice site mutation. None of the heterozygotes had suffered from significant bleeding. The mutation C2362F and the splice site mutation in intron 13 are commonly observed in subjects with recessive von Willebrand disease in the Veneto region. These mutations are truly recessive and cause bleeding only in the compound heterozygous or homozygous state. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: von Willebrand disease; von Willebrand factor; Inherited bleeding disorder
1. Introduction von Willebrand disease (VWD) is an inherited bleeding disorder characterized by a quantitative or qualitative defect of von Willebrand factor (VWF), an adhesive protein involved in platelet adhesion and which acts as a carrier of FVIII in blood [1]. The disease has been classified into three major groups, based on phenotypic criteria: Types 1 and 3 represent, respectively, partial and severe quantitative deficiency of VWF whereas Type 2 includes qualitative defects [1]. The molecular bases of Type 3 VWD are heterogeneous [1]. The probands may be homozygotes for
* Corresponding author. Tel.: +39-444-993679; fax: +39-444-993922. E-mail address: [email protected] (G. Castaman).
null alleles or compound heterozygotes of null alleles and missense mutations, especially when low levels of VWF are detectable [1– 3]. In some cases, it is difficult to distinguish Type 3 from recessive Type 1 on phenotypic grounds [2,3]. In a group of eight patients with recessive VWD from the Veneto region, the propositus of a single family showed compound heterozygosity for a missense mutation in exon 42 (G7085T leading to C2362F substitution in the mature subunit) and a splice site mutation C ! A at position 11/24 in the consensus sequence of intron 13 [3]. Both mutations were observed in other families with either Type 1 or Type 3 phenotype in combination with other null alleles [3]. Heterozygous subjects were asymptomatic, as usually reported for the obligatory heterozygotes of Type 3 VWD [3]. In this paper, we report a new family with autosomal recessive VWD in which the propositus was compound heterozygous for the C2362F mutation and the splice site
0049-3848/02/$ – see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 0 0 7 - 5
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mutation in intron 13. Haplotype analysis confirmed the possibility of a founder effect for both the mutations.
2. Materials and methods
were expressed as international units (IU), with reference to an internal plasma pool calibrated against the Second International Reference Preparation for Factor VIII/VWFrelated activities in plasma. Bleeding time (BT) was measured with the Simplate II device (General Diagnostics, Morris Plains, NJ).
2.1. Case report 2.3. DNA studies The propositus, born in 1993, was referred for investigation at age 3 after an episode of epistaxis requiring substitution treatment with FVIII/VWF concentrate. At age 2, he bled profusely after an accidental bite to the tongue. After the diagnosis, he suffered from several episodes of epistaxis, controlled by oral tranexamic acid and/or digital compression. He also had frequent easy bruising. At another hospital, factor VIII procoagulant activity (FVIII:C) was 25% and von Willebrand factor antigen (VWF:Ag) 5%. All the available family members were investigated. No bleeding episodes were observed in the relatives. The pedigree is shown in Fig. 1. 2.2. Coagulation studies Blood was anticoagulated with 3.8% sodium citrate (1:10 vol:vol) and centrifuged at 2500 g for 15 min. FVIII:C, VWF:Ag and VWF ristocetin cofactor activity (VWF:RCo) in plasma and platelets were determined as previously described [4]. All FVIII and VWF measurements
2.3.1. Haplotype analysis Genomic DNA was extracted from peripheral blood leukocytes and amplified by the polymerase chain reaction (PCR) [3] under the following conditions: denaturing at 94 °C for 4 min followed by 32 cycles of 1-min denaturing at 94 °C; 1-min annealing at 58 °C; and 2 min extension at 72 °C. Haplotypes were determined using two variable number tandem repeat (VNTR) polymorphisms located in intron 40 (indicated as VNTR I and VNTR II), RsaI polymorphism in exon 13 (G+/A 1451, Arg/His 484), RsaI polymorphism in exon 18 (A+/G 2365, Thr/Ala 789), an HphI polymorphism in exon 28 (G /A+ 4141, Ala/Thr 1381) and XmnI polymorphism in exon 45 (+ indicates presence, absence of the restriction site; nucleotide numbering from the A of the initiator ATG as + 1; amino acid numbering from the translation initiation codon), as previously described in detail [3]. VNTR I and II were examined by electrophoresis on 8% nondenaturing polyacrylamide gels and visualized under
Fig. 1. Pedigree of the family and haplotypes of the propositus and his parents. The indicated haplotypes are VNTR I and II intron 40, RsaI exon 13, RsaI exon 18, HphI exon 28, and XmnI exon 45, respectively (see Materials and Methods). The proband is indicated by an arrowhead. ND = not done.
G. Castaman et al. / Thrombosis Research 105 (2002) 135–138 Table 1 Von Willebrand factor gene mutations screened in the propositus Mutation
Exon
Method of detectiona
Splice site intron 13 4449delG 7125insC C2362F R2535a C2671Y
14 28 42 42 45 49
Eco57I sequence analysis sequence analysis Eco57I DdeI MaeIII
a
For details see Ref. [3].
UV light following ethidium bromide staining. The restriction enzyme analyses were performed directly in the PCR reaction mixture. The samples were incubated overnight at 37 °C. 2.3.2. Mutation screening DNA was amplified by PCR using oligonucleotide primers designed on the basis of the reported sequences of the VWF gene [5]. The propositus was screened for the presence of several mutations (Table 1), which were previously identified in our group of patients with recessive VWD from Veneto [3]. Digestion with the appropriate restriction enzyme was carried out directly in the PCR reaction mixture. The samples were incubated overnight at 37 °C. The pattern of digestion was visualized under UV after ethidium-bromide-stained agarose gel electrophoresis. 3. Results The propositus, subject III.2, showed marked reduction of all FVIII/VWF measurements, compared with the other
137
family members investigated (Table 2). Platelet VWF was severely reduced (VWF:Ag 2.8 IU/10 11 platelets and VWF:RCo < 3 IU/1011 platelets; normal range 17 –88 and 24 – 102 IU/1011 platelets, respectively). The BT was markedly prolonged. The father, subject II.4, showed only borderline values of VWF:RCo in plasma, whereas platelet VWF was in the normal range (not shown). The mother, subject II.5, had all FVIII/VWF measurements mildly reduced in plasma and only platelet VWF:RCo slightly reduced in platelets (18 IU/1011 platelets). BT was normal in the parents as well as in all the other family members tested. Factor VIII/VWF measurements showed a heterogeneous pattern in the remaining subjects, with subject II.6 having all the measurements reduced and the other seven with only mild VWF reduction. Only subject I.1 with the splice site mutation had normal FVIII/VWF measurements. The propositus (subject III.2; Table 2) was compound heterozygous for the splice site mutation in intron 13, inherited from the father, and the C2362F mutation, inherited from the mother (Fig. 1). All other screened mutations (Table 1) were not detected in the propositus. Apart from the propositus, five members were heterozygous for the splice site mutation and six for the C2362F mutation (Fig. 1). None of these true heterozygotes had suffered from bleeding symptoms. Haplotype analysis showed that the splice site mutation was linked to the haplotype 7, 4, +, +, , + (VNTR I and VNTR II intron 40, RsaI exon 13, RsaI exon 18, HphI exon 28 and XmnI exon 45, respectively), and the C2362F mutation to the haplotype 10, 4, +, +, , (Fig. 1). These haplotypes were similar to those previously found in association with these mutations in our population of patients with recessive VWD.
Table 2 FVIII and VWF measurements, BT, blood group, and presence of the C2362F or intron 13 splice site mutation Subjects
VIII:C (IU/dl)
VWF: Ag (IU/dl)
VWF:RCo (IU/dl)
BT (min)
Blood group
VWF gene mutation
I.1 I.2 I.3 I.4 II.1 II.2 II.4 II.5 II.6 II.7 II.8 III.1 III.2 (propositus) III.3 III.4 III.5 III.6 III.7 III.8 Normal range
149 140 128 95 116 101 66 – 102 35 38 100 120 102 18 – 21 85 82 87 114 104 126 52 – 173
109 120 123 56 53 98 70 – 77 25 34 102 117 80 5–7 45 47 35 58 56 117 47 – 165
73 130 132 41 52 97 58 – 76 29 28 80 140 78 <3–7 42 49 48 61 55 118 51 – 188
5 ND ND 4.5 6 ND 7 4 6 5.5 4.5 ND >15 ND ND 5 ND ND ND < 7.5
O B A O O O B O O A A O B O O O A A A –
splice site NO NO C2362F splice site NO splice site C2362F C2362F NO NO NO C2362F/splice site splice site splice site C2362F C2362F C2362F NO NONE
ND = not done.
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4. Discussion We have identified a new family with recessive VWD in which the propositus was compound heterozygous for a splice site mutation in intron 13 and the C2362F mutation in exon 42 of VWF gene. While these mutations have been previously identified in a propositus with recessive Type 3 VWD, this patient was not related to the present family [3]. When compared to the previous case, the present propositus had somewhat higher FVIII/VWF parameters in plasma and apparently milder clinical phenotype, so that a Type 1 VWD phenotype could be appreciated. There is no clear explanation for this finding. The only difference between the two propositi is blood group (Type B in the present, Type O in the previously reported case). However, in keeping with possible diagnosis of Type 3 VWD, platelet VWF was severely reduced in the present propositus. Each mutation was linked to a similar haplotype as in the families previously reported [3]. This strongly suggests the presence of a founder effect. As a further support to this hypothesis, these mutations have not been reported from other geographical areas. The splice site mutation leads to aberrant mRNA splicing and skipping of exon 14 [3]. Consequently, only a nonfunctional truncated VWF is coded for by this null allele. The C2362F mutation induces the loss of a cysteine in a VWF region to which no function has been assigned. Patients homozygous or hemizygous for the C2362F mutation show an increased proteolysis of the mutant C2362F VWF [6], as usually observed in typical Type 2A VWD patients [7]. In contrast, heterozygotes show a normal pattern, suggesting a protective role of the VWF coded for by the normal allele. Interestingly, patients with compound heterozygosity for this mutation and a null allele have a significant VIII:C increase after desmopressin, despite the fact that very little VWF increase is observed after infusion and BT remains markedly prolonged [4]. Heterozygosity for either the C2362F or the splice site mutation in intron 13 does not represent a risk factor for bleeding. In fact, using the pooled data from the present analysis and from the previous investigation [3], none of the
subjects who are heterozygous for C2362F mutation (n = 15) or for the splice site mutation (n = 9) suffered from bleeding, despite the occurrence of several hemostatic challenges. These observations are in keeping with the typical description of an absence of bleeding symptoms in obligatory heterozygotes for Type 3 VWD [8]. On the contrary, we observed a single patient with homozygosity for C2362F mutation and a moderate to severe bleeding history [3]. In conclusion, the C2362F mutation and the splice site mutation in intron 13 of VWF gene are commonly observed in our population of patients with recessive VWD in the Veneto region (northeast of Italy) and are likely to be the result of a founder effect.
References [1] Nichols WC, Ginsburg D. von Willebrand disease. Medicine 1997; 76:1 – 20. [2] Eikenboom JCJ, Reitsma PH, Peerlinck KMJ, Brie¨t E. Recessive inheritance of von Willebrand’s disease type I. Lancet 1993;341:982 – 6. [3] Eikenboom JCJ, Castaman G, Vos HL, Bertina RM, Rodeghiero F. Characterization of the genetic defects in recessive type 1 and type 3 von Willebrand disease patients of Italian origin. Thromb Haemost 1998;79:709 – 17. [4] Castaman G, Lattuada A, Mannucci PM, Rodeghiero F. Factor VIII:C increases after desmopressin in a subgroup of patients with autosomal recessive von Willebrand disease. Br J Haematol 1995;89:147 – 51. [5] Mancuso DJ, Tuley EA, Westfield LA, Worrall NK, Shelton-Inloes BB, Sorace JM, Alevy YG, Sadler JE. Structure of the gene for human von Willebrand factor. J Biol Chem 1989;264:19514 – 27. [6] Castaman G, Eikenboom JCJ, Lattuada A, Mannucci PM, Rodeghiero F. Heightened proteolysis of the von Willebrand factor subunit in patients with von Willebrand disease hemizygous or homozygous for the C2362F mutation. Br J Haematol 2000;108:188 – 90. [7] Zimmerman TS, Dent JA, Ruggeri ZM, Nannini LH. Subunit composition of plasma von Willebrand factor. Cleavage is present in normal individuals, increased in type IIA and IIB von Willebrand disease, but minimal in variants with aberrant structure of individuals oligomers (types IIC, IID, and IIE). J Clin Invest 1986;77:947 – 51. [8] Mannucci PM, Lattuada A, Castaman G, Lombardi R, Colibretti ML, Ciavarella N, Rodeghiero F. Heterogeneous phenotypes of platelet and plasma von Willebrand factor in obligatory heterozygotes for severe von Willebrand’s disease. Blood 1989;74:2433 – 7.